This invention is generally in the area of methods for the treatment of viral diseases, and more specifically relates to the treatment of AIDS using thiazine dyes.
Methylene blue, 3,7-Bis(dimethylamino)phenothiazin-5-ium chloride, C16H18ClN3S, is a dark green or blue thiazine dye which was first isolated in 1876. It is FDA approved for oral administration and has been reported to be effective as an antiseptic, disinfectant, and antidote for cyanide and nitrate poisoning. For over 50 years it has been known that methylene blue is reduced by mitochondria to leukodye which is then auto-oxidized back to methylene blue by oxygen, yielding H2O2. This is the probable mechanism by which methylene blue, injected i.v. at a dose of 1 mg/kg body weight, is effective in the treatment of methemoglobinemia, a clinical disorder where more than 1% of the hemoglobin in the blood has been oxidized to FE3+. Kelner and Alexander reported in J. Biol. Chem. 260(28), 15168-15171 (1985), that methylene blue oxidizes glutathione directly when it is reduced by NADPH, rather than via the H2O2.
Methylene blue, in the presence of light, has been reported to damage DNA, probably by destroying or cleaving the DNA at the guanine residues. Simon and Van Vunakis, Arch. Biochem. Biophys. 105, 197-206 (1964), noted that the effect of several photoactive dyes and light is dependent on the concentration of the dye, as well as light wavelength and intensity, and can be correlated with uptake of oxygen and decrease in ultraviolet absorbance by guanine derivatives. Kornhauser, et al., Photochem. Photobiol. 18, 63-69 (1973) attempted to characterize the changes in guanosine following exposure to methylene blue and light using thin layer chromatographic analytical techniques.
Others have also attempted, without success, to analyze the actual mechanism of the effect of methylene blue and light on DNA. Friedman and Brown, Nucleic Acids Res. 5, 615-622 (1978), showed that methylene blue and light caused lesions at deoxyguanosines in DNA and that subsequent exposure to piperidine caused strand rupture. They hypothesized that cyclo-addition occurred at various positions in the purine ring, rendering the DNA susceptible to base catalyzed cleavage following modification of the other nucleoside bases.
Waskell, et al., reported in Biochim. Biophys. Acta 129, 49-53 (1966), that extensive irradiation of polynucleotides in the presence of methylene blue causes extensive destruction of guanosine, leaving ribose, guanidine, ribosylurea, and free urea. They postulated that the destruction of the guanosine residues was the mechanism for a previous observation by Sastry, et al., Biochim. Biophys. Acta, 129, 42 (1966), that, in vitro, methylene blue and irradiation inactivates Tobacco Mosaic Virus (TMV) RNA, rendering the virus uninfective. Singer and Fraenkel-Conrat, have also reported, in Biochem. 4, 2446-2450 (1966), that another type of dye, thiopyronin (where the ring N is replaced by CH), and proflavin, cause inactivation of TMV RNA in the presence of light. This is also the probable mechanism for the observation that topical administration of a 0.1% solution of methylene blue in conjunction with polychromatic light photoinactivates viruses such as herpes simplex, as referenced in American Hospital Formulary Service 92:00 Unclassified Therapeutic Agents, page 2176 editor, Gerald K. McEvoy (American Society of Hospital Pharmacists, Inc. 1981 revised 1988). Other observations have been made showing in vitro inactivation of viruses using light, methylene blue, and electricity, as reported in J. Clin.Microbiol., 17(2), 374-376 (1983), by Badylak, et al., (pseudorabies virus) and Proc.Soc.Exper.Biol.Med. 161, 204-209 (1979) by Swartz, et al., (Herpes simplex).
U.S. Ser. No. 264,088 entitled xe2x80x9cPhototherapy Using Methylene Bluexe2x80x9d filed Oct. 28, 1988 by Robert A. Floyd disclosed a method for using thiazine dyes, especially methylene blue, in combination with light to hydroxylate guanosine or deoxyguanosine at the C8 of the purine ring. The number of guanosines in a nucleic acid strand converted to 8xe2x80x94OH-deoxyguanosine (8xe2x80x94OH-dG) or 8xe2x80x94OH-guanosine (8xe2x80x94OH-G) can be controlled through manipulation of the concentration of methylene blue, light intensity and length of exposure, pH, and buffer strength. Very little, if any, other derivatives are formed. As described, the method can be used for the selective mutation or modification of either a DNA or a RNA sequence, or the protein expressed therefrom. 8xe2x80x94OH-dG and 8xe2x80x94OH-G do not base pair well and are especially susceptible to misreading.
Hydroxylation of guanine in DNA to produce 8-hydroxydeoxyguanosine (8xe2x80x94OH-dG) has been postulated to be an important factor in mutation and carcinogenesis by Kasai and Nishimura, Nucleic Acid Res. 12, 2137-2145 (1984); Gann. 75, 565-566 and 841-844 (1984); Environ. Health Perspect. 67, 111-116 (1986); Kasai, et al., Gann. 75, 1037-1039 (1984); Carcinogenesis 7, 1849-1851 (1986); Aida and Nishimura, Mutation Res. 192, 83-89 (1987). Kuchino, et al., Nature (London) 327, 77-79 (1987) used synthetic oligonucleotides containing 8-hydroxydeoxyguanosine in a specific position as a template for DNA synthesis to show misreading at the modified base and at adjacent pyrimidine bases. They observed that specific base-pairing was completely lacking at the 8-hydroxyguanosine and that incorrect bases were inserted at the adjacent pyrimidine bases. Kasai, et al., reported in Carcinogenesis 8(12), 1959-1961 (1987) that administration of a renal carcinogen, potassium bromate, to the rat caused a significant increase of 8-hydroxydeoxyguanosine in the kidney DNA, but not in non-target organ DNA.
Chemically, 8-hydroxydeoxyguanosine is made from guanosine by the action of reagents which generate oxygen radicals, such as ascorbic acid and other reducing agents, metals, polyphenols, and asbestos, and by x-irradiation. Intracellular DNA appears to undergo repair by enzymes following formation of 8-hydroxy-deoxyguanosine. This may be a naturally occurring response which has evolved to combat the effects of the many mutagens, tumor promoters, and carcinogens which cause the formation of 8-hydroxydeoxyguanosine.
As described in U.S. Ser. No. 264,088 issued as U.S. Pat. No. 4,950,665 on Aug. 21, 1990, the selective administration of thiazine dyes can be used in the treatment of viral infections and in cancer. Selective delivery can be achieved using systems such as liposomes for delivery to macrophages and other phagocytic cells or biodegradable controlled release implants. Viruses, bacteria, and cells undergoing rapid DNA synthesis are all inactivated by methylene blue when irradiated with light. Treatment can be extracorporeal or by light irradiation of specific tissues using other methods. Methylene blue absorbs in the red wavelengths, i.e., approximately 670 nm, which penetrates tissue much better than other lower wavlengths.
Acquired Immunodeficiency Syndrome (AIDS) is generally accepted at this time to be a consequence of infection with the retrovirus variously termed human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), AIDS associated retrovirus (ARV), or human immunodeficiency virus (HIV-1). There is considerable difficulty in diagnosing the risk of development of AIDS. AIDS is known to develop in at least 50% of the individuals infected with human immunodeficiency virus (HIV), although this percentage is suspected to be much higher.
A patient is generally diagnosed as having AIDS when a previously healthy adult with an intact immune system acquires impaired T-cell immunity. The impaired immunity usually appears over a period of eighteen months to three years. As a result of this impaired immunity, the patient becomes susceptible to opportunistic infections, various types of cancer such as Kaposi""s sarcoma, and other disorders associated with reduced functioning of the immune system.
No treatment capable of preventing or curing HIV infection is currently available, although several compounds have demonstrated antiviral activity against the virus in vitro, including HPA-23, interferons, ribavirin, phosphonoformate, ansamycin, suramin, imuthiol, penicillamine, rifabutin, AL-721, 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT), and other 2xe2x80x2,3xe2x80x2-dideoxynucleosides. AZT is the only drug which has been demonstrated to prolong life of patients infected with HIV. However, AZT is quite toxic when used for periods of several months and must be discontinued even in those patients initially tolerant to the drug due to the drug causing severe anemia. See Yarchoan et al., Lancet, 575-580 (1986). Further, AZT-resistant strains of HIV have now been reported in patients undergoing treatment with AZT, Larder, B. A., Darby G., Richmond, D. D., Science 243, 1731-1734 (1989).
Many inhibitors of cellular processes, such as AZT, limit viral replication, but are at the same time quite toxic for the host as well. Most of the antiviral drugs that have been discovered so far cannot be prescribed for a prolonged period of time because of their toxicity. Accordingly, it is clear that there is a strong need for new antiviral agents, especially those with low toxicity to normal cells. More particularly, because of the high mortality of AIDS and the lack of an effective treatment for this disease, there remains a great need for development of new low toxicity agents for prophylactic use as well as long term therapy of AIDS patients.
It is therefore an object of the present invention to provide methods and compositions for treatment or prevention of viral infections.
It is further object of the present invention to provide methods and compositions for selectively inactivating HIV.
A method for using thiazine dyes, especially methylene blue, alone or in combination with low levels of light, to selectively inactivate or inhibit intracellular replication of specific viruses, especially human immunodeficiency virus.
Examples of useful thiazine dyes are methylene blue, azure A, azure C, toluidine blue O, and thionine. The preferred dye at this time is methylene blue. Methylene blue is FDA approved for topical, i.v., and oral administration, and has minimal side effects. Selective delivery can be achieved using systems such as liposomes for delivery to macrophages and other phagocytic cells or using biodegradable controlled release implants. Since methylene blue absorbs in the red wavelengths, i.e., approximately 670 nm, which penetrates tissue much better than other lower wavelengths, light penetrating the skin to the capillaries at the surface can be used to enhance the activity of the dye.